U.S. patent application number 10/218109 was filed with the patent office on 2003-04-10 for therapeutic agents for achondroplasia.
Invention is credited to Nakao, Kazuwa.
Application Number | 20030068313 10/218109 |
Document ID | / |
Family ID | 26623312 |
Filed Date | 2003-04-10 |
United States Patent
Application |
20030068313 |
Kind Code |
A1 |
Nakao, Kazuwa |
April 10, 2003 |
Therapeutic agents for achondroplasia
Abstract
The present invention aims to provide novel therapeutic agents
for achondroplasia caused by mutations in FGFR3. Therapeutic agents
for achondroplasia caused by the cartilage growth inhibition
resulting from mutations in the gene for fibroblast growth factor
receptor 3 (FGFR3), comprising a substance activating guanylyl
cyclase B (GC-B) as an active ingredient are disclosed.
Inventors: |
Nakao, Kazuwa; (Kyoto-shi,
JP) |
Correspondence
Address: |
HUNTON & WILLIAMS
INTELLECTUAL PROPERTY DEPARTMENT
1900 K STREET, N.W.
SUITE 1200
WASHINGTON
DC
20006-1109
US
|
Family ID: |
26623312 |
Appl. No.: |
10/218109 |
Filed: |
August 14, 2002 |
Current U.S.
Class: |
424/94.61 ;
514/17.1; 514/9.1 |
Current CPC
Class: |
A61P 19/08 20180101;
A61K 38/2242 20130101; A61P 5/00 20180101; A61P 3/00 20180101 |
Class at
Publication: |
424/94.61 ;
514/12 |
International
Class: |
A61K 038/47; A61K
038/18 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 28, 2001 |
JP |
301586/2001 |
Oct 5, 2001 |
JP |
310322/2001 |
Claims
What is claimed is:
1. A therapeutic agent for achondroplasia caused by the cartilage
growth inhibition resulting from mutations in the gene for
fibroblast growth factor receptor 3 (FGFR3), comprising a substance
activating guanylyl cyclase B (GC-B) as an active ingredient.
2. The agent of claim 1 wherein the cartilage growth inhibition is
rescued by enlarging hypertrophic chondrocytes and increasing the
extracellular matrix of the proliferative chondrocyte layer.
3. The agent of claim 1 or 2 wherein the substance activating GC-B
is a peptide.
4. The agent of claim 3 wherein the peptide is a C-type natriuretic
peptide (CNP).
5. The agent of claim 4 wherein the CNP is CNP-22 or CNP-53.
Description
BACKGROUND OF THE INVENTION
[0001] (i) Field of the Invention
[0002] The present invention relates to agents and methods for
treating achondroplasia.
[0003] (ii) Description of the Related Art
[0004] Achondroplasia is one of the most common congenital diseases
responsible for micromelic dwarfism characterized by short limbs
relative to trunk. It is diagnosed by X-ray photographs in addition
to growth failure in the major axes of the long bones of
extremities and typical physical features such as a large frontally
projecting cranium and a short nose. The disease is said to occur
at an incidence of one to 10,000-25,000 people. This disease is an
autosomal dominant hereditary disorder, but 80-90% of cases are
found to be sporadic. Current therapies include orthopedic
surgeries such as artificial hip joint replacement or leg
lengthening and growth hormone therapy. Leg lengthening involves
cutting bones at the age of 10 years or after and gradually
increasing body height using a special device (leg lengthening
device) over several courses of about six months. However, this
procedure inflicts a great pain on patients. Growth hormone therapy
increases body height by means of periodic growth hormone
injections starting from childhood. However, growth ceases when
injections are stopped. Neither therapy is curative, and neither
are considered ideal from the viewpoint of patients' QOL (American
Journal of Medical Genetics 72: 71-76, 1997; European Journal of
Endocrinology 138: 275-280, 1998). Consequently, it is desirable to
develop a achondroplasia therapy based on a new mechanism.
[0005] Recent reports show that achondroplastic patients have
mutations in fibroblast growth factor receptor 3 (FGFR3) located at
chromosome 4p16.3, and two mutations are currently known. Of these
mutations, 97% represents G1138A (change of 1138th G to A) and 2.5%
represents G1138C (change of 1138th G to C), resulting in a change
of the amino acid Gly at the 380-position to Arg (G380R) (Nature
371: 252-254, 1994; Cell 78: 335-342, 1994). To examine the
relation of this mutation to achondroplasia, G380R FGFR3 (sometimes
hereinafter referred to as FGFR3.sup.ach) transgenic mice were bred
to provide an animal model for human achondroplasia. The mice
showed short limbs and craniofacial hypoplasia (Development. 125:
4977-4988, 1998).
[0006] On the other hand, the natriuretic peptide (NP) family
consists of three peptides, ANP (atrial natriuretic peptide), BNP
(brain natriuretic peptide) and CNP (C-type natriuretic peptide),
and is thought to show biological activity by increasing
intracellular cGMP through two guanylyl cyclase coupled receptors
(GC-A receptor for ANP and BNP, and GC-B receptor for CNP) (Annu.
Rev. Biochem. 60: 229-255, 1991). NPs are reported to have
important roles in the regulation of body fluid homeostasis and
blood pressure control (J. Clin. Invest. 93: 1911-1921, 1987; J.
Clin. Invest. 87: 1402-1412, 1994), but also they are known by
their expression and physiological activity in various tissues
other than cardiovascular system (Endocrinology. 129: 1104-1106,
1991; Annu. Rev. Biochem. 60: 553-575, 1991). Among them, they have
a role as bone growth factor. In organ cultures of tibiae from
fetal mice, CNP significantly promotes longitudinal bone growth (J.
Biol. Chem. 273: 11695-11700, 1998). CNP is more potent than ANP
and BNP in the production of cGMP in organ cultures of tibiae from
fetal mice, cultured chondrocytes and cultured osteoblasts (J.
Biol. Chem. 269: 10729-10733, 1994; Biochem. Biophys. Res. Commun.
223: 1-6, 1996; Biochem. Biophys. Res. Commun. 215: 1104-1110,
1995). CNP and its receptor GC-B are expressed in the growth plates
of bones (J. Biol. Chem. 273: 11695-11700, 1998; Proc. Natl. Acad.
Sci. U.S.A. 95: 2337-2342, 1998). CNP was also found to have a role
in thickening the cartilage layer of the growth plate in transgenic
mice expressing CNP specifically in cartilage (Yasoda et al.,
Abstracts of the 72nd meeting of the Japan Endocrinology Society,
1999).
[0007] The relation of CNP to dwarfism was also indicated because
CNP knockout mice developed dwarfism (Proc. Natl. Acad. Sci. U.S.A.
98: 4016-4021, 2001), but nothing has been described about its
relation to achondroplasia caused by FGFR3 mutations and no
positive evidence has shown that CNP is effective for
achondroplasia caused by FGFR3 mutations. That is, it is known that
FGFR3 mutations are related with achondroplasia and that CNP is
involved in chondrogenesis, but nothing has been known so far about
the relation between them, particularly which of FGFR3 and CNP is
located upstream in the regulatory pathway of endochondral
ossification and whether or not CNP has a therapeutic effect for
achondroplasia.
[0008] An object of the present invention is to provide novel
agents and methods for treating achondroplasia caused by mutations
in FGFR3.
SUMMARY OF THE INVENTION
[0009] On the hypothesis that a substance (e.g., CNP) activating
guanylyl cyclase B (GC-B) may be applied to diseases involving
chondrogenesis, we searched for a suitable achondroplasia model and
mated this animal model with CNP-transgenic mice to prepare double
transgenic mice for testing whether the symptoms of achondroplasia
can be corrected. As described above, G380R FGFR3 (FGFR3.sup.ach)
transgenic mice had been bred as an animal model of human
achondroplasia, which showed short limbs and craniofacial
hypoplasia (Development. 125: 4977-4988, 1998). Thus, we obtained
such FGFR3.sup.ach-transgenic mice and mated them with our
CNP-transgenic mice to prepare CNP/FGFR3.sup.ach-double transgenic
mice, which were found to remedy the bone growth inhibition caused
by FGFR3.sup.ach, whereby we achieved the present invention
relating to agents and methods for treating achondroplasia with
CNP.
[0010] Accordingly, the present invention provides therapeutic
agents for achondroplasia caused by the cartilage growth inhibition
resulting from mutations in the gene for fibroblast growth factor
receptor 3 (FGFR3), containing a substance activating guanylyl
cyclase B (GC-B) as an active ingredient, as well as methods for
treating achondroplasia comprising administering a substance
activating guanylyl cyclase B (GC-B).
[0011] As used herein, the expression "achondroplasia caused by the
cartilage growth inhibition resulting from mutations in the gene
for fibroblast growth factor receptor 3 (FGFR3)" means
achondroplasia caused by hyperactivity or function control failure
of FGFR3 or overexpression of the FGFR3 gene resulting from
mutations in the FGFR3 gene, and achondroplasia is synonymous with
chondrogenesis disorder. As used herein, FGFR3.sup.ach means
fibroblast growth factor receptor 3 (FGFR3) containing a mutation
of the amino acid Gly at the 380-position substituted to Arg
(G380R), which is known to induce hyperactivity of FGFR3
(Development. 125: 4977-4988, 1998).
[0012] As used herein, the expression "substance activating
guanylyl cyclase B" means a substance (peptide or low molecular
compound) capable of binding to GC-B known as a receptor for CNP
(C-type natriuretic peptide) to activate it, preferably a substance
(peptide or low molecular compound) having CNP (C-type natriuretic
peptide)-like activity, such as mammalian CNP (CNP-22 (Biochem.
Biophys. Res. Commun. 168: 863-870, 1990, WO91/16342), CNP-53
(Biochem. Biophys. Res. Commun. 170: 973-979, 1990, JPA 1992-74198,
JPA 1992-139199), avian CNP (JPA 1992-120094), amphibian CNP (JPA
1992-120095) and CNP analog peptides (JPA 1994-9688), preferably
mammalian CNP, more preferably CNP-22. Identification of the
"substance activating guanylyl cyclase B" is performed by, for
example, expressing GC-B receptor in cultured cells such as COS-7,
incubating the medium with a candidate substance (peptide or low
molecular compound) at a given temperature for a given period
(e.g., 37.degree. C., 5 min) and then determining the concentration
of cGMP in the cell extracts (Science 252: 120-123, 1991).
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 shows generation of transgenic mice that overexpress
CNP specifically in cartilage. A: Schematic representation showing
the structure of a recombinant gene for generating CNP-transgenic
mice. B: Photograph showing the results of Southern hybridization
using the tail DNA of CNP-transgenic mice. C: Photographs showing
the results of RT-PCR analysis of the expression of Col II-CNP in
various organs from CNP-transgenic mice.
[0014] FIG. 2 shows the appearance of CNP-transgenic mice. A:
Photographs showing the skeletons of a nontransgenic mouse (upper)
and a CNP-transgenic mouse (lower) at the age of 1 day. B: Graphs
showing the growth curves of male (left) and female (right)
CNP-transgenic mice including heterozygotes (closed circles) and
homozycotes (closed squares) as compared with nontransgenic
littermates (open circles). C: The left panel shows soft x-ray
photographs of the cranium (upper) and the lower extremities
(lower) of 6-mo-old female nontransgenic littermates (left) and
female CNP-transgenic mice (right), and the right panel shows a
graph showing comparison of the length of some bones of
nontransgenic female littermates (open bar) and female
CNP-transgenic mice (closed bar) measured from the photographs on
the left panel.
[0015] FIG. 3 shows histological analysis of the growth plate of
CNP-transgenic mice. A-D: Photographs showing Alcian blue and
hematoxylin/eosin staining (3-wk-old), A: tibial growth plate of
nontransgenic littermates (x50), B: tibial growth plate of
CNP-transgenic mice (x50), C: tibial growth plate of nontransgenic
littermates (x200), D: tibial growth plate of CNP-transgenic mice
(x200). E-H: Photographs showing in situ hybridization analysis
with collagen cDNA probes (2-wk-old), E: tibial growth plate of
nontransgenic littermates (type II collagen, x200), F: tibial
growth plate of CNP-transgenic mice (type II collagen, x200), G:
tibial growth plate of nontransgenic littermates (type X collagen,
x200), H: tibial growth plate of CNP-transgenic mice (type X
collagen, x200). I-J: Photographs showing von Kossa staining
(3-wk-old), I: epiphyseal trabecular bones of nontransgenic
littermates (x50), J: epiphyseal trabecular bones of CNP-transgenic
mice (x50). X-L: Photographs showing Brdurd staining (2-wk-old), K:
tibial growth plate of nontransgenic littermates (x50), L: tibial
growth plate of CNP-transgenic mice (x50).
[0016] FIG. 4 shows organ culture of the tibiae of CNP-transgenic
mice. A: The left panel shows photographs showing the appearance of
the tibiae of 16.5-d fetal mice after 4-d culture from
nontransgenic littermates (upper left), CNP-transgenic mice (upper
right), nontransgenic littermates in the medium containing HS-142-1
(50 mg/L) (lower left) and CNP-transgenic mice in the medium
containing HS-142-1 (50 mg/L) (lower right). The right panel shows
a graph showing the time course of the growth of the length of the
tibiae from the start to the end of 4-d culture. Open circles:
nontransgenic littermates, n=6; open squares: CNP-transgenic mice,
n=6; closed circles: nontransgenic littermates (HS-142-1), n=6;
closed squares: CNP-transgenic mice (HS-142-1), n=6. *P<0.05
CNP-transgenic mice versus their nontransgenic littermates,
**P<0.05 HS-142-1-treated nontransgenic littermates versus
untreated nontransgenic littermates, ***P<0.01 HS-142-1-treated
CNP-transgenic mice versus untreated CNP-transgenic mice. B: Graph
showing the cGMP content of the cultured tibiae of the fetal
CNP-transgenic mice (n=5). *P<0.01 CNP-transgenic mice versus
their nontransgenic littermates. C: Graph showing .sup.35SO.sub.4
incorporation into the cultured tibiae of the fetal CNP-transgenic
mice (n=6). *P<0.05 CNP-transgenic mice versus their
nontransgenic littermates.
[0017] FIG. 5 shows photographs showing histochemical analysis of
the cultured tibiae of CNP-transgenic mice (Alcian blue and
hematoxylin/eosin staining). A: nontransgenic littermates (x25); B:
CNP-transgenic mice (x25); C: CNP-transgenic mice (treated with
HS-142-1) (x25); D: nontransgenic littermates (x200); E:
CNP-transgenic mice (x200); F: CNP-transgenic mice (treated with
HS-142-1) (x200).
[0018] FIG. 6 shows gross phenotypes of CNP-transgenic,
FGFR3.sup.ach-transgenic and CNP/FGFR3.sup.ach-double transgenic
mice. A: Photographs showing the gross appearance of 3-mo-old
nontransgenic littermate, CNP-transgenic mice,
FGFR3.sup.ach-transgenic mice and CNP/FGFR3.sup.ach-double
transgenic mice from top to bottom. B: Graph showing the growth
curves of the naso-anal length of female FGFR3.sup.ach-transgenic
mice (closed triangles), female CNP/FGFR3.sup.ach-transgenic mice
(open squares) and nontransgenic littermates (closed circles)
(n=7). C: Photographs showing detection of the expression of Col
II-CNP by RT-PCR using total RNA from the cartilage of
nontransgenic littermates (lane 1), CNP-transgenic mice (lane 2)
and FGFR3.sup.ach-transgenic mice (lane 3). D: Left panel shows
photographs showing the appearance of the skeleton of 3-mo-old
nontransgenic littermates, CNP-transgenic mice,
FGFR3.sup.ach-transgenic mice and CNP/FGFR3.sup.ach-double
transgenic mice from top to bottom. Right panel shows a graph
showing comparison of the length of various bones of nontransgenic
littermates (open bar), CNP-transgenic mice (closed bar),
FGFR3.sup.ach-transgenic mice (hatched bar) and
CNP/FGFR3.sup.ach-double transgenic mice (shaded bar) (n=4).
*P<0.05. The lengths of cranium (naso-occipital), cranium
(width), humerus, femur and vertebra are shown.
[0019] FIG. 7 shows photographs showing histochemical analysis of
the tibial growth plate from 2-wk-old mice (Alcian blue and
hematoxylin/eosin staining). A: nontransgenic littermates (x50); B:
FGFR3.sup.ach-transgeni- c mice (x50); C:
CNP/FGFR3.sup.ach-transgenic mice (x50); D: nontransgenic
littermates (x100); E: FGFR3.sup.ach-transgenic mice (x100); F:
CNP/FGFR3.sup.ach-transgenic mice (x100).
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION
[0020] The CNP-transgenic mice prepared by us increased their body
length with longitudinal overgrowth of bones through endochondral
ossification. Further analysis of the CNP-transgenic mice by
histochemical analysis of the growth plate showed 1) increased
thickness of the growth plate along with the elongation of both
proliferative and hypertrophic chondrocyte layers, 2) enlarged
extracellular matrix in the proliferative chondrocyte layer, and 3)
increased size of the mature hypertrophic chondrocytes. These facts
show that CNP promotes the expression of the differentiation trait
of chondrocytes in each differentiation stage of the growth plate,
rather than contributes to the commitment to the differentiation or
proliferation of chondrocytes of the growth plate, along with the
fact that no appreciable alteration in the proliferation of
chondrocytes was observed as assayed by BrdUrd staining in the
hypertrophic chondrocyte layers of the growth plate of the
CNP-transgenic mice. This is supported by the fact that the
expression of type X collagen mRNA in the hypertrophic chondrocytes
in the growth plate of the CNP-transgenic mice had an intensity
comparable to that of their nontransgenic littermates though the
expression cell area enlarged. However, the width of the cranium,
which is made through membranous ossification, was not changed in
the CNP-transgenic mice. This suggests that CNP is not expressed in
the cranium, or is not involved in the process of membranous
ossification.
[0021] Ex vivo organ culture experiments provided further
information about the action mechanism of CNP in the growth plate.
The elongation of the cartilagenous primordia with enlarged
extracellular matrix and increased size of hypertrophic
chondrocytes in cultured tibiae from CNP-transgenic mice was
potent, as obtained in cultured tibiae from their nontransgenic
littermates in the presence of 10.sup.-7 M CNP. This histological
change was completely abolished by adding a non-peptide NP receptor
antagonist HS-142-1 (Circ. Res. 78: 606-614, 1996), like the case
when HS-142-1 was added to cultured tibiae from their nontransgenic
littermates incubated with 10.sup.-7 M CNP. These results show that
the Col II-CNP transgene (the gene containing a mouse CNP cDNA
fragment inserted into a DNA segment of the mouse procollagen al
type II (Col 2a1) promoter region as described in Example 1)
functions well to alter the phenotype in vivo in the growth plate
cartilage, along with the fact that the production of the second
messenger of CNP, cGMP, increases in cultured tibiae from
CNP-transgenic mice. The increase in the synthesis of the
extracellular matrix, as shown by the increase of .sup.35S
incorporation in cultured tibiae from CNP-transgenic mice, is
compatible with the enlargement of the extracellular matrix in the
growth plate of CNP-transgenic mice. This can explain the
elongation mechanism of the growth plate in CNP-transgenic mice.
The elongation of metaphyseal cancellous bone observed in
CNP-transgenic mice indicates that the replacement of cartilage to
calcified bone was proceeded smoothly. These experiments revealed
the importance of CNP in endochondral ossification.
[0022] Next, we obtained G380R FGFR3 (FGFR3.sup.ach)-transgenic
mice (from Professor David M. Ornitz of Washington University, US)
and mated them with CNP-transgenic mice to prepare
CNP/FGFR3.sup.ach-double transgenic mice. In
CNP/FGFR3.sup.ach-double transgenic mice, both CNP-Tg gene and
FGFR3.sup.ach-Tg gene are expressed in the resting chondrocyte
layer and proliferative chondrocyte layer of the growth plate and
the symptoms of dwarfism of FGFR3.sup.ach-transgenic mice were
visibly improved. The endogenous CNP, GC-B and FGFR3 were expressed
in the proliferative chondrocyte layer and the prehypertrophic
chondrocyte layer.
[0023] The effect of the present invention is best shown in FIG. 6.
FIG. 6A shows the gross appearance of 3-mo-old nontransgenic
littermates, CNP-transgenic mice, FGFR3.sup.ach-transgenic mice and
CNP/FGFR3.sup.ach-double transgenic mice from top to bottom, and
FIG. 6D shows their skeletal appearance. The naso-anal length of
CNP/FGFR3.sup.ach-double transgenic mice is almost comparable to
that of nontransgenic littermates, showing that the shortening of
the length of the limbes observed in FGFR3.sup.ach-transgenic mice
can be rescued by the overexpression of CNP.
[0024] The fact that CNP improved the symptoms of dwarfism of
FGFR3.sup.ach-transgenic mice suggests that CNP is not, at least in
most part, located upstream of FGFR3 in the regulatory pathway of
endochondral ossification. The shortened growth plate in
FGFR3.sup.ach-transgenic mice was elongated by the overexpression
of CNP in both proliferative and hypertrophic chondrocyte layers,
but some histological features were different from those of the
nontransgenic littermates. The extracellular matrices of both
proliferative and hypertrophic chondrocyte layers enlarged so that
the alignment of hypertrophic chondrocytes was disordered or
hypertrophic chondrocytes enlarged. Considering that overexpressed
CNP did not affect the delayed formation of the secondary
ossification center in FGFR3.sup.ach-transgenic mice, CNP does not
seem to be involved in the commitment to the differentiation of
chondrocytes as FRFR3 does, but rather seems to promote the gene
expression of chondrocytes in each differentiation stage. That is,
the pathway in which CNP regulates endochondral ossification may be
different from that of FGFR3.
[0025] Further in vitro study of the interaction between CNP and
FGFR3 using a mouse chondrocyte strain showed that CNP/GC-B systems
and basic FGF/FGFR3 systems (basic FGF is a ligand for FGFR3)
together influence intracellular transmission of information in
chondrocytes.
[0026] Without being bound to the specific theory described above,
we confirmed from the results described above that the growth
retardation of FGFR3.sup.ach-transgenic mice is rescued by the
overexpression of CNP though CNP and FGFR3 have different
regulatory mechanisms of endochondral ossification. This suggested
that CNP has a therapeutic effect as a drug for promoting the
growth of long bones with the purpose of treating achondroplastic
patients, whereby the present invention was achieved. A major known
cause of achondroplasia is hyperactivity of FGFR3 resulting from
mutations in the FGFR3 gene, but achondroplasic symptoms may also
be caused by function control failure of FGFR3 and enhanced
expression of the FGFR3 gene. A novel therapy can be provided for
these achondroplasic symptoms by activating GC-B or promoting the
gene expression, protein expression and protein function of its
ligand CNP. To promote the gene expression of CNP, the expression
of the endogenous CNP gene may be enhanced or gene therapy may also
be applied by transferring an exogenous CNP gene into the living
body.
[0027] Therapeutic agents for achondroplasia of the present
invention are prepared from a substance activating GC-B as an
active ingredient in combination with a carrier or exipient and
other additives used for ordinary formulation.
[0028] Suitable carriers and excipients for formulation include,
for example, lactose, magnesium stearate, starch, talc, gelatin,
agar, pectin, acacia, olive oil, sesame oil, cacao butter, ethylene
glycol and other common additives.
[0029] Suitable solid compositions for oral administration include
tablets, pills, capsules, powders and granules. In such solid
compositions, at least one active ingredient is mixed with at least
one inert diluent, such as lactose, mannitol, glucose,
hydroxypropylcellulose, microcrystalline cellulose, starch,
polyvinylpyrrolidone, or magnesium aluminometasilicate. The
compositions may conventionally contain additives other than inert
diluents, e.g., lubricants such as magnesium stearate,
disintegrants such as calcium carboxymethylcellulose, and
solubilizers such as glutamic acid or aspartic acid. Tablets or
pills may, if desired, be coated with a sugar coating or a gastric
or enteric film comprising sucrose, gelatin, hydroxypropyl
methylcellulose phthalate or the like or may be coated with two or
more layers. Capsules of an absorbable material such as gelatin are
also included.
[0030] Liquid compositions for oral administration include
pharmaceutically acceptable emulsions, solutions, suspensions,
syrups and elixirs, and may contain ordinary inert diluents, such
as purified water and ethanol. In addition to inert diluents, these
compositions may contain adjuvants such as wetting agents or
suspending agents, sweetening agents, flavoring agents, aromatics
and preservatives.
[0031] Injections for parenteral administration include sterile
aqueous or nonaqueous solutions, suspensions and emulsions. Aqueous
solutions and suspensions contain water for injection and
physiological saline for injection, for example. Nonaqueous
solutions and suspensions contain propylene glycol, polyethylene
glycol, vegetable oils such as olive oil, alcohols such as ethanol,
and POLYSORBATE 80 (registered trademark). These compositions may
further contain adjuvants, such as preservatives, wetting agents,
emulsifying agents, dispersing agents, stabilizers (e.g., lactose),
and solubilizers (e.g., glutamic acid and aspartic acid). These can
be sterilized by ordinary sterilizing methods, such as mechanical
sterilization with a microfiltration membrane, heat sterilization
such as autoclaving or inclusion of a bactericide. Injections may
be solution formulations or freeze-dried formulations to be
reconstituted before use. Suitable excipients for freeze-drying
include, for example, sugar alcohols and sugars such as mannitol or
glucose.
[0032] When therapeutic agents of the present invention are used
for gene therapy, they may contain a substance activating GC-B such
as a CNP-related nucleic acid integrated downstream of a promoter
sequence that is functional in host cells such as Cytomegalovirus
promoter (CMV promoter) in a virus vector, preferably a lentivirus
vector, an adeno-associated virus vector, more preferably an
adenovirus vector, or in a known vehicle suitable for gene therapy
such as a chemically synthesized liposome, a virus envelope or a
complex of a virus envelop and a chemical liposome.
[0033] Therapeutic agents for achondroplasia of the present
invention are preferably administered via pharmaceutically common
routes such as oral or parenteral routes. When the active
ingredient is a GC-B agonist antibody, they are normally
administered via parenteral routes such as injection (subcutaneous,
intravenous, intramuscular or intraperitonial injection) or
percutaneous, mucosal, nasal or pulmonary administration, but may
also be orally administered.
[0034] The amount of the substance activating GC-B contained as an
active ingredient in formulations of the present invention can be
determined depending on the type of disease to be treated, the
severity of the disease, the age of the patient and other factors,
but generally can be administered in the range of 0.005
.mu.g/kg-100 mg/kg, preferably 0.025 .mu.g/kg-5 mg/kg.
[0035] Therapeutic agents for achondroplasia of the present
invention can be used in combination with conventional therapies
such as growth hormones or orthopedic surgeries such as artificial
hip joint replacement or leg lengthening.
[0036] The present invention includes, but is not limited to, the
following aspects.
[0037] (1) A therapeutic agent for achondroplasia caused by the
cartilage growth inhibition resulting from mutations in the gene
for fibroblast growth factor receptor 3 (FGFR3), containing a
substance activating guanylyl cyclase B (GC-B) as an active
ingredient.
[0038] (2) The agent as defined in (1) above wherein the cartilage
growth inhibition is rescued by enlarging hypertrophic chondrocytes
and increasing the extracellular matrix of the proliferative
chondrocyte layer.
[0039] (3) The agent as defined in (1) or (2) above wherein the
substance activating GC-B is a peptide.
[0040] (4) The agent as defined in (3) above wherein the peptide is
a C-type natriuretic peptide (CNP).
[0041] (5) The agent as defined in (4) above wherein the CNP is
CNP-22 or CNP-53.
[0042] (6) The agent as defined in (1) or (2) above wherein the
substance activating GC-B is a low molecular compound.
[0043] (7) The agent as defined in (1) or (2) above wherein the
agent containing a substance activating GC-B as an active
ingredient promotes the gene expression, protein expression or
protein function of the substance activating GC-B.
[0044] (8) The agent as defined in (1) or (2) above wherein the
agent containing a substance activating GC-B as an active
ingredient promotes the expression of a gene for CNP, the
expression of a CNP protein or the function of a CNP protein.
[0045] (9) A method for treating achondroplasia caused by the
cartilage growth inhibition resulting from mutations in the gene
for fibroblast growth factor receptor 3 (FGFR3), comprising
administering a substance activating guanylyl cyclase B (GC-B).
[0046] (10) The method as defined in (9) above, comprising rescuing
the cartilage growth inhibition by enlarging hypertrophic
chondrocytes and increasing the extracellular matrix of the
proliferative chondrocyte layer.
[0047] (11) The method as defined in (9) or (10) above wherein the
substance activating GC-B is a peptide.
[0048] (12) The method as defined in (11) above wherein the peptide
is a C-type natriuretic peptide (CNP).
[0049] (13) The method as defined in (12) above wherein the CNP is
CNP-22 or CNP-53.
[0050] (14) The method as defined in (9) or (10) above wherein the
substance activating GC-B is a gene (for example, DNA) encoding a
peptide.
[0051] (15) The method as defined in (14) above wherein the peptide
is a C-type natriuretic peptide (CNP).
[0052] (16) The method as defined in (15) above wherein the CNP is
CNP-22 or CNP-53.
[0053] (17) The method as defined in any one of (14) to (16) above,
comprising transferring a gene encoding a peptide directly or in a
vector (for example, adenovirus-derived vector) or a liposome
suitable for gene therapy.
[0054] (18) A use of the substance as defined in any one of (3) to
(6) above for preparing a therapeutic agent for achondroplasia
caused by the cartilage growth inhibition resulting from mutations
in the gene for fibroblast growth factor receptor 3 (FGFR3)
[0055] The following examples further illustrate the present
invention.
EXAMPLES
Example 1
Preparation of a Recombinant Gene for Generating CNP-Transgenic
Mice
[0056] As shown in FIG. 1A, a mouse CNP cDNA fragment encoding
amino acid residues 1-127 (489 bp; FEBS Lett. 276: 209-213, 1990)
was inserted into a DNA segment of the mouse procollagen al type II
(Col 2a1) promoter region (6.5 kb; Dev. Dyn. 204: 202-210, 1995).
This promoter region DNA segment was supplied from B. de
Crombrugghe, M. D. Anderson Cancer Center, Huston. This promoter
region DNA segment containing a promoter, exon 1, intron 1 and an
artificial splice acceptor site was fused to the downstream CNP
cDNA fragment. The initiation codon in exon 1 of this promoter
region DNA segment was inactivated by point mutagenesis. A DNA
segment (0.3 kb) containing a bovine growth hormone polyadenylation
signal was added to the downstream of the CNP cDNA. The NotI/NotI
DNA fragment (7.3 kb) as shown in FIG. 1A was purified for
injection into fertilized oocytes and used as a col-CNP DNA
solution.
Example 2
Generation of CNP-Transgenic Mice
[0057] The mice used for collecting fertilized eggs to be
microinjected with the col-CNP DNA solution (hereinafter referred
to as injecting DNA solution) were C57BL/6J inbred mice purchased
from CLEA Japan, Inc. (egg collecting mice). Females at 8 weeks of
age or older were superovulated and mated with males at 8 weeks of
age or older to collect many fertilized eggs, which were
transferred to M2 medium and cultured in a 5% carbon dioxide
incubator at 37.degree. C. Then, 2 pL of the injecting DNA solution
was injected into the male pronucleus of each of said fertilized
eggs by microinjection using a DNA injection pipette. The
fertilized eggs injected with the injecting DNA solution were
transferred to M16 medium and cultured overnight in a 5% carbon
dioxide incubator at 37.degree. C. The female mice used for
pregnancy, delivery and nursing of offspring from the fertilized
eggs injected with the injecting DNA solution (foster mother mice)
and the male mice mated with the females were ICR inbred mice
purchased from CLEA Japan, Inc. Vasoligated male mice at 8 weeks of
age or older were mated with female mice at 8 weeks of age or
older, among which those showing a vaginal plug were used as foster
mothers. The left and right oviducts of each foster mother were
exposed by surgery using an anesthetic intraperitoneally injected
at 0.01 ml/g body weight containing Nembutal (Dainabot Co., Ltd.,
50 mg/mL sodium pentobarbital) diluted to 12% in a diluent (a mixed
solution of 20 mL propylene glycol, 10 mL ethanol and 70 mL
sterilized water). Among the fertilized eggs cultured overnight,
those having developed into 2-cell embryos were collected and 10-15
of them were inserted into each oviduct, after which the incised
site was sutured. Foster mothers were raised for 3 weeks and if
they delivered, the tail of each offspring was dissected at about 1
cm 5 weeks after birth to isolate and purify chromosomal DNA using
Easy-DNA Kit (Invitrogen). This tail DNA was checked for the
presence of the transgene by PCR. The mice in which the presence of
the transgene was confirmed were reared as founder transgenic mice
up to the age of 7 weeks and then naturally mated with
nontransgenic C57BL/6J at 7 weeks of age or older to give
transgenic progeny.
[0058] The gene microinjection experiment yielded 5278 eggs from a
total of 336 egg-collecting mice C57BL/6J, and the injecting DNA
solution was injected into 2280 eggs identified as fertilized eggs
among them. On the following day, 1600 eggs (70%) developed into
2-cell embryos, 1476 of which were implanted into the oviducts of a
total of 60 foster mothers. Thirty-seven foster mothers became
pregnant and gave birth to a total of 108 offspring (7%). An assay
for the transgene by PCR in the tail DNA showed that a total of 4
founder transgenic mice (4%) (2 males, 2 females) were obtained.
These founder transgenic mice were naturally mated with
nontransgenic C57BL/6J to give progeny in which the transgene was
transmitted in two strains (male Tg-1055, female Tg-1077).
Example 3
Genetic Analysis of CNP-Transgenic Mice
3-1 Verification of Gene Transfer into Transgenic Mice by PCR
[0059] The transgene was verified by Southern hybridization using
the isolated and purified tail DNA. The tail DNA was digested with
a restriction enzyme SacI and subjected to Southern hybridization
with a .sup.32P-labeled CNP cDNA fragment (526 bp) to give a 2.1 kb
band for the transgene and a 3.0 kb band for the endogenous gene
(FIG. 1B). The copy number was assessed by comparing the strength
of the 2.1 kb band with the strength of the 3.0 kb endogenous band,
and the male strain Tg-1055 shown to contain 10 copies was used for
further analysis.
3-2 Expression Analysis of the IskD77N Gene by PCR
[0060] Expression analysis of the transgene was performed by the
Real Time-PCR method. Cartilage from the lower vertebra and the
tail and other organs were rapidly dissected from newborn
nontransgenic and transgenic mice and stored in liquid nitrogen.
They were homogenized by a Physcotoron homogenizer (NITION Medical
Supply, Chiba, Japan) and then, total RNA was isolated and purified
with an ISOGEN reagent. A Superscript first strand synthesis kit
(GIBCO/BRL, Gaithersburg, MD) was used to synthesize cDNA with
oligo-dT primers, and PCR was then performed using the forward
primer (in exon 1) and reverse primer (in cDNA) as shown in FIG.
1A. The PCR reaction involved 45 cycles of a three-step reaction
consisting of 95.degree. C. for 30 seconds, 58.degree. C. for 30
seconds and 72.degree. C. for 1 minutes. After the PCR reaction, a
10 .mu.L aliquot was assayed by electrophoresis on 1% agarose gel.
The 450-bp positive band was detected only in cartilage, but not in
brain, heart, lung, liver, kidney, intestine and muscle. The 450-bp
positive band was not detected in the cartilage and other organs of
their nontransgenic littermates.
Example 4
Determination of the Growth Curve of CNP-Transgenic Mice
[0061] The length between the nose to the anus (hereinafter
referred to as naso-anal length) was measured every week to draw a
growth curve of mice. At the perinatal stage, CNP-transgenic mice
and their nontransgenic littermates were not distinguished from
each other. At 1 day after birth, Alzarin red S and Alcian blue
staining of bones and cartilage revealed longitudinal overgrowth of
both bones and cartilage in CNP-transgenic mice, including long
bones of limbs, vertebrae and skulls (FIG. 2A). No delay in the
ossification was observed in the periphery of limbs at this stage.
Ossification centers of phalanges had already appeared in
CNP-transgenic mice as well as their nontransgenic littermates. As
they grew, CNP-transgenic mice gradually showed a prominent
increase in the naso-anal length (FIG. 2B). Female 10-wk-old
CNP-transgenic mice were 19% longer than their female nontransgenic
littermates (n=7). Male CNP-transgenic mice were longer than their
male nontransgenic littermates (n=7), but to an extent lower than
female mice (10%). Homozygous CNP-transgenic male mice were longer
than heterozygous CNP-transgenic male mice (female 6%, male 4%,
n=7). Soft X-ray analysis showed a significant increase in 6-mo-old
CNP-transgenic mice as compared with their nontransgenic
littermates in the length of limbs, vertebrae and the longitudinal
axis of the skull, all of which were formed by endochondral
ossification, although the width of the cranium did not increase
(FIG. 2C). The increase was especially prominent in vertebrae and
proximal long bones (humerus and femur), which were longer by 28%,
25% and 23% (n=6) than those of their nontransgenic littermates,
respectively.
Example 5
Histological Analysis of CNP-Transgenic Mice
[0062] For light microscopy, the tibiae and vertebrae were removed
and fixed in 10% formalin/PBS (pH 7.4). The calcified bones were
demineralized in 10% formalin/PBS (pH 7.4) containing 20% EDTA.
Paraffin blocks were prepared by standard histological procedures.
Sections (5-6 .mu.m) were prepared at several levels and stained
with Alcian blue (pH 2.5) and then counterstained with
hematoxylin/eosin. The length of the layers of the growth plate,
the diameter of the matured hypertrophic chondrocytes and the
BrdUrd labeling index in the proliferative chondrocyte layer were
analyzed on a Macintosh computer using an NIH Image program. For
BrdUrd staining, 2-wk-old mice were intraperitoneally injected with
BrdUrd (100 .mu.g/g body weight) and killed after 1 h.
Immunohistochemical staining of incorporated BrdUrd in cells in the
growth plate of the tibiae was performed by standard methods. To
evaluate the mineralized stage of each sample, Von Kossa staining
was done on undecalcified sections.
[0063] For in situ hybridization analysis, digoxigenin-labeled
sense and antisense riboprobes were prepared from a rat pro-a1(X)
collagen cDNA fragment and a mouse pro-a1 (II) collagen cDNA
fragment by using a digoxigenin RNA labeling kit (Roche
Diagnostics, Indianapolis, Ind.).
[0064] No typical histological change in the epiphyseal cartilage
was found in CNP-transgenic mice at the prenatal stage, but as they
grew, the height of the growth plate of long bones of the vetebrae
of CNP-transgenic mice significantly increased at least at the age
of 3 weeks or after (FIGS. 3A, B). Among the growth plate cartilage
layers of the tibiae of 3-wk-old mice, both hypertrophic
chondrocyte layer (234.+-.12 .mu.m versus 207.+-.14 .mu.m, n=4,
p<0.05) and proliferative chondrocyte layer (215.+-.3 .mu.m
versus 193.+-.16 .mu.m, n=4, p<0.05) of CNP-transgenic mice were
longer than those of nontransgenic littermates. The hypertrophic
chondrocyte layer and proliferative chondrocyte layer were shown to
express type X collagen or type II collagen by in situ
hybridization analysis (FIGS. 3 E-H). Higher magnification revealed
an increase of the size of chondrocytes (24.3.+-.1.2 .mu.m versus
21.2.+-.1.3 .mu.m, n=6, p<0.05) (FIGS. 3 C, D). The length of
the resting chondrocyte layer was not changed even in
CNP-transgenic mice. The band of BrdUrd positive chondrocytes was
widened in CNP-transgenic mice relative to their nontransgenic
littermates, though the number of BrdUrd positive chondrocytes was
comparable (13.3.+-.3% versus 12.5.+-.2.9%, n=4) (FIGS. 3 K, L).
Von Kossa staining of the growth plate of the tibiae of 3-wk-old
mice revealed that the epiphyseal trabecular bones formed by
adjacent hypertrophic chondrocyte layer were obviously longer, and
the volume of the trabecular bones was larger in CNP-transgenic
mice than in their nontransgenic littermates (FIGS. 3 I, J).
Example 6
Effects of the Cartilage-Specific Expression of CNP on Cultured
Embryonic Tibiae from CNP-Transgenic Mice
[0065] Tibiae from the fetus of CNP-transgenic mice or their
nontransgenic littermates were dissected out on 16.5-d post coitus
and cultured for 4 days in suspension in an artificial medium. To
inhibit the effect of the endogenous CNP, the tibial culture was
performed with a non-peptide NP receptor antagonist, HS-142-1
(Komatsu et al., Circ Res. 78:606-614, 1996) at a concentration of
50 mg/L in the medium. At the end of the culture period, the
cultured tibiae were measured for their longitudinal length, and
fixed and embedded for histological analysis. Sections of 5 .mu.m
in thickness were cut from the embedded specimen and stained with
Alcian blue (pH 2.5) and counterstained with hematoxylin/eosin. The
cGMP contents of the cultured tibiae were measured by RIA at the
end of the 4-d culture period. Glycosaminoglycan synthesis of the
cultured tibiae was assessed by measuring .sup.35SO.sub.4
incorporation (Mericq et al., Pediatr Res 47: 189-193, 2000).
Namely, cultured tibiae of the CNP-transgenic mice and their
nontransgenic littermates were labeled with 5 .mu.Ci/ml
Na.sub.2.sup.35SO.sub.4 (Amersham, specific activity 100 mCi/mmol)
for 1 h. The cultured tibiae were then rinsed three times for 10
min with Pack's saline (Sigma Chemical Co., St. Louis, Mo.), and
then digested in 1.5 ml of fresh medium containing 0.3% papain for
24 h at 60.degree. C. Then, the culture was incubated with 0.5 ml
of 10% cetylpyridinium chloride (Sigma Chemical Co.)--0.2 M NaCl at
room temperature for 18 h to precipitate glycosaminoglycan. The
precipitate was washed three times with 1 ml of 0.1%
cetylpyridinium chloride (Sigma Chemical Co.)--0.2 M NaCl and then
dissolved in 1 ml of 23 N formic acid, after which the
.sup.35SO.sub.4 content was determined by a liquid scintillation
counter.
[0066] Even before incubation, the tibial explants from
CNP-transgenic mice were significantly longer than those from their
nontransgenic littermates (FIG. 4A). During incubation, the tibial
explants from CNP-transgenic mice increased prominently in
longitudinal length and were about 35% longer than those from
nontransgenic littermates at the end of the 4-d culture (n=6, FIG.
4A). The increase of the cartilagenous primordium was prominent
(40% increase) among all parts of the tibial explant. HS-142-1
known to inhibit the effect of the endogenous CNP in cartilage
could inhibit spontaneous growth of the tibial explants from
nontransgenic littermates (FIG. 4A). Moreover, the increase in the
length of the tibial explants from CNP-transgenic mice was
completely abolished by HS-142-1 (50 mg/L) to the extent of the
length of the tibiae from nontransgenic littermates treated with
HS-142-1 (FIG. 4A). The content of cGMP in the cultured tibiae from
CNP-transgenic mice was about 9 times higher than that in tibiae
from nontransgenic littermates (18.7.+-.1.2 fmol/mg protein versus
2.1.+-.0.2 fmol/mg protein, n=5, FIG. 4B). Glycosaminoglycan
synthesis was about 25% increased in tibiae from CNP-transgenic
mice compared with those from their nontransgenic littermates
(2300.+-.170 cpm/ tibia versus 1840.+-.140 cpm/ tibia, n=6, FIG.
4C). Histologically, the epiphyseal cartilage of the tibial
explants from CNP-transgenic mice increased in the height of both
proliferative chondrocyte layer (369.+-.26 .mu.m versus 287.+-.14
.mu.m, n=4, p<0.05) and hypertrophic chondrocyte layer
(450.+-.29 .mu.m versus 294.+-.16 .mu.m, n=4, p<0.05), with the
increased extracellular space stained by Alcian blue as
cartilagenous matrix in the proliferative chondrocyte layer (FIGS.
5A, B). Also, the hypertrophic chondrocyte layer enlarged
(17.8.+-.0.8 .mu.m versus 15.4.+-.1.4 .mu.m, n=6, p<0.05, FIG.
5D). Alteration induced by HS-142-1 at the same dose in the
epiphyseal cartilage of the cultured tibiae from CNP-transgenic
mice also disappeared.
Example 7
Analysis of CNP/FGFR3.sup.ach-Double Transgenic Mice
[0067] Female CNP-transgenic mice and male FGFR3.sup.ach-transgenic
mice (obtained from Professor David M. Ornitz of Washington
University, US) were mated. As FGFR3.sup.ach-transgenic mice were
originally produced on the FVB/N background, only F1 double
transgenic mice were used in contrast to their CNP, FGFR3.sup.ach
and nontransgenic littermates.
[0068] At the age of 3 months, CNP-transgenic mice were longer than
their nontransgenic littermates and FGFR3.sup.ach-transgenic mice
were shorter than their nontransgenic littermates (FIG. 6A). The
naso-anal length of CNP/FGFR3.sup.ach-transgenic mice was almost
comparable to that of the nontransgenic littermates. The CNP
expression level in the cartilage of CNP/FGFR3.sup.ach-transgenic
mice was comparable to the expression level in CNP-transgenic mice
(FIG. 6C). The growth curve of the naso-anal length of
CNP/FGFR3.sup.ach-transgenic mice, FGFR3.sup.ach-transgenic mice
and their nontransgenic littermates showed that the growth
retardation in FGFR3.sup.ach-transgenic mice was rescued by
overexpression of CNP in the growth plate cartilage. At the age of
10 weeks, the naso-anal length of CNP/FGFR3.sup.ach-transgenic mice
was 94.7.+-.4.0 mm, which was 8% longer than that of
FGFR3.sup.ach-transgenic mice (87.7.+-.2.6 mm) and comparable to
that of their nontransgenic littermates (97.0.+-.4.2 mm) (FIG. 6B).
Soft X-ray analysis revealed that the shortening of the length of
the bones observed in FGFR3.sup.ach-transgenic mice, including the
naso-occipital length of the cranium and the longitudinal length of
the humerus, femur and vertebrae (L1-7), was also partially rescued
in CNP/FGFR3.sup.ach-transgenic mice. The width of the cranium was
not affected in either FGFR3.sup.ach-transgenic or
CNP/FGFR3.sup.ach-transgenic mice (FIG. 6D). The microscopic
analysis of the growth plate cartilage of the proximal tibiae from
2-wk-old CNP/FGFR3.sup.ach-transgenic mice,
FGFR3.sup.ach-transgenic mice and their nontransgenic littermates
showed that the height of the hypertrophic chondrocyte layer of
FGFR3.sup.ach-transgenic mice decreased as compared with that of
nontransgenic littermates (169.+-.15 .mu.m versus 220.+-.15 .mu.m).
It was recovered in CNP/FGFR3.sup.ach-transgenic (229.+-.21 .mu.m,
FIGS. 7A-C). However, the disordered alignment of the column of the
hypertrophic chondrocytes and the enlarged extracellular matrix in
the prehypertrophic and upper hypertrophic chondrocyte layers were
observed in CNP/FGFR3.sup.ach-transgenic mice in contrast to
FGFR3.sup.ach-transgenic mice and their nontransgenic littermates
(FIGS. 7D-F). The size of each hypertrophic chondrocyte in
CNP/FGFR3.sup.ach-transgenic mice was significantly larger than
that of FGFR3.sup.ach-transgenic mice and their nontransgenic
littermates (20.1.+-.1.5 .mu.m, 18.4.+-.1.2 .mu.m, 19.0.+-.0.2
.mu.m, n=6, p<0.05, FIG. 7D-F). In the proximal tibiae of
10-wk-old mice, the secondary ossification center was not formed
yet in FGFR3.sup.ach-transgenic mice and
CNP/FGFR3.sup.ach-transgenic mice, whereas that was well organized
in their nontransgenic littermates (FIGS. 7A-C).
Example 8
Study on the Interaction Between CNP and FGFR3 Using a Mouse
Chondrocyte Strain
[0069] Cells of the mouse chondrocyte strain ATDC (J. Bone. Miner.
Res., 12, 1174-1188, 1997; supplied from Assistant Professor
Shukunami and Professor Hiraki of the Institute for Frontier
Medical Sciences, Kyoto University) were pretreated with 1-10 ng/ml
basic FGF (SIGMA), a ligand for FGFR3. Then, these cells were
stimulated with 10.sup.-9-10.sup.-7 M CNP and assayed for
intracellular cGMP production by the RIA method (cyclic GMP Assay
Kit available from YAMASA CORPORATION). Phosphorylation of p44 and
p42 MAP kinases (ERK1/2) and expression of MAP kinase (MEK) and p44
MAP kinase (ERK1) after basic FGF stimulation were also assayed by
Western blotting using phosphorylated MAP-K antibodies and MAP-K
antibodies (both available from Cell Signaling Technology; MAP:
mitogen-activated protein).
[0070] The results showed that intracellular cGMP production after
CNP stimulation following pretreatment with 1 ng/ml basic FGF for 1
h decreased to 70% of control. Phosphorylation of ERK1/2 with basic
FGF by pretreatment for 1 h was significantly inhibited by
10.sup.-7 CNP.
[0071] This revealed that CNP/GC-B systems and basic FGF/FGFR3
systems together influence intracellular transmission of
information in chondrocytes.
[0072] Advantages of the Invention
[0073] Therapeutic agents for achondroplasia provided by the
present invention can treat achondroplasia by acting as a gene for
CNP, a CNP protein or a low molecular compound activating GC-B on a
site other than directed by growth hormones. Therapeutic agents for
achondroplasia of the present invention can offer an excellent
therapy with improved QOL of patients by relieving burden and pain
on the patients as compared with conventional orthopedic surgeries
such as artificial hip joint replacement or leg lengthening.
Moreover, transgenic animals disclosed herein can be used to test
their efficacy against achondroplasia caused by mutations other
than G380R in FGFR3.
* * * * *